Ion cyclotron resonance mass spectrometers (ICR-MS), also known as Fourier transform mass spectrometers (FTMS) or, in full, as Fourier transform ion cyclotron resonance mass spectrometers (FTICR-MS), are at present the mass spectrometers that offer the highest mass resolution and the most accurate measurement of mass. In these spectrometers, the ions are excited into cyclotron movement under ultrahigh vacuum conditions in a very intense magnetic field of seven, nine, twelve or even fifteen Tesla, generated by superconducting coils held at the temperature of liquid helium, and the frequency of these circulating movements of the ions is measured. The frequencies are inversely proportional to the masses of the ions. Since the magnetic field generated by the superconducting coils is extraordinarily stable, and since frequency measurements are amongst the most accurate measurements that can be taken by today's physical technology, the masses of the circulating ions can be determined with greater accuracy than by any other type of mass spectrometer.
Unfortunately, this ion cyclotron frequency is shifted by the space charge that is created by the ions in the measuring cell of the ICR-MS. A “reduced cyclotron frequency” is measured, which is non-linearly dependent on the strength of the space charge. This has been known for a long time. The publication by J. B. Jeffries, S. E. Barlow and G. H. Dunn, International Journal of Mass Spectrometry and Ion Processes 54, 169-187, (1983) gives a theoretical description of the frequency shift as a consequence of space charge. If the space charge varies from one scan to the next because it is not regulated, it can cause a shift in the mass signal that differs every time.
At higher ion densities, a further undesirable phenomenon, known as “peak coalescence”, occurs in ICR mass spectrometry. Signals from ions whose masses only differ very slightly converge, and in extreme cases the ion signals may even completely merge. The result of this merging is, in most cases, another high-resolution ion signal that wrongly indicates an apparent mass lying between the true masses of the two ion species. The analysis of ion signals that lie very close together is, however, a task that ICR mass spectrometers are often called upon to perform.
Any kind of frequency shift will result in incorrect mass measurements, and must therefore be avoided. Control methods for filling the measuring cell of an ICR-MS are therefore described in patent specification U.S. Pat. No. 6,555,814 B1 (G. Baykut, J. Franzen). The control methods described there, however, always refer to the measurement of the total ion current (or of a fixed proportion of the total ion current) alone, with the result that the regulation of the filling level is always focused on the total ion charge that has been inserted into the measuring cell. Experience, however, shows that maintaining a constant charge quantity does not protect against various kinds of non-reproducible frequency shifts. In addition to the total charge, the precise composition of the mixture of ions of different mass and charge also plays a role, as is already clear from the phenomenon of ion signal merging that has been mentioned above.
Electrospray ionization is nowadays the most widely used ionization method for the ICR mass spectrometry of biomolecules. In this method, ions are generated out of the solution of the analyte molecules at atmospheric pressure under high voltage (3-6 kV) between an electrospray needle and a counter-electrode. Although the spraying procedure is often supported by a slow, finely controllable spray pump (or by a liquid chromatography feed pump, known by the acronym HPLC), the driving force of the spraying method is the detachment of small, charged droplets resulting from a high ion density on the liquid surface (Coulomb repulsion) under the influence of a powerful electrical field. A “dry gas” that flows in the direction opposite to that of the flight of the charged droplets causes the solvent to evaporate from the droplets (the desolvation process), therefore causing the droplet radii to diminish. As a result of the Coulomb forces that have been strengthened in this way, ionized molecules are evaporated, in most cases in multiply protonated form, i.e. as positively charged ions. These ions are fed for measurement to the mass spectrometer through an inlet capillary, a multi-stage vacuum system and a multipole ion guide.
Electrospray ionization under atmospheric pressure has made it very easy to couple separation methods for dissolved analyte substances, such as liquid chromatography or capillary electrophoresis, directly to the mass spectrometer. Ionization by laser desorption (LDI) has for a long time been used successfully to transfer large organic molecules from a solid surface into the gaseous phase, and thereby to ionize them. A special type of LDI is ionization by matrix-assisted laser desorption (MALDI). MALDI involves the analyte molecules being mixed with what is known as a matrix substance. The ratio of analyte to matrix molecules here is typically between 1:102 and 1:104. The laser beam is absorbed by matrix molecules; in the process, a portion of this matrix material evaporates, taking analyte molecules with it into the gaseous phase. The process partially ionizes them. In most cases the ionization occurs by proton acceptance. Substances used as a matrix are most often proton donors, i.e. substances that easily give up protons.